The objectives of the proposed research are to define the relationship between molecular structure and catalytic function in complex enzymes and to elucidate the evolutionary pathways and mechanisms that have led to the attainment of end product regulation by specific branch point enzymes in metabolism. Parallel studies of two enzymes of aromatic amino acid biosynthesis will be undertaken. These are the aroH DAHP (deoxyarobinoheptulosonate-phosphate) synthase of Escherichia coli, which is one of the three isoenzymes that catalyze the first specific step of the common aromatic amino acid pathway, and the trpE subunit of the anthranilate synthase of Salmonella typhimurium, which catalyzes the first specific step of the tryptophan pathway. Although catalytically distinct, both enzymes are specifically regulated by the same feedback inhibitor, L-tryptophan. Molecular genetic, biochemical and immunological methodologies will be employed to analyze the aroH and trpE genes and polypeptides. The catalytic and regulatory domains of the proteins will be identified and mapped by mutagenesis and DNA sequence analysis of cloned aroH and trpE genes, and, in the case of aroH, by the exchange of domains between it and the tyrosine-sensitive (aroF) and the phenylalanine-sensitive (aroG) DAHP synthase isozymes. These studies should reveal whether the tryptophan-binding domains of the trpE and aroH polypeptides are homologous in sequence or structure, whether they might be of common evolutionary origin, and whether domain recruitment has served as a mechanism for the evolution of feedback-inhibitable enzymes.